1. Field of the Invention
The present invention relates to preparations of proinsulin C-peptide. The preparations are in the form of a gel and allow for sustained release of C-peptide over a length of time. Such preparations may be used in the treatment of diabetes and/or diabetic complications.
2. Description of Related Art
Insulin-dependent diabetes mellitus (IDDM), generally synonymous with type 1 diabetes, is the classical, life-threatening form of diabetes, the treatment of which was revolutionized by the discovery of insulin in 1922. The prevalence of IDDM is unfortunately widespread throughout much of the world and hence IDDM represents a serious condition with a significant drain on health resources.
The etiology of IDDM is multifactorial and not yet entirely clear. However it is characterised by a partial or complete destruction of the pancreatic beta cells. In the acute phase of IDDM insulin deficiency it is thus the dominating pathophysiological feature.
After starting insulin treatment many patients enjoy good blood glucose control with only small doses of insulin. There is an early phase, the “honeymoon period”, which may last a few months to a year and which probably reflects a partial recovery of beta cell function. This is, however, a temporary stage and ultimately, the progressive destruction of the beta cells leads to complete cessation of insulin secretion and increasing requirements for exogenous insulin.
While the short term effects of hypoinsulinemia in the acute phase of IDDM can be well controlled by insulin administration, the long term natural history of IDDM is darkened by the appearance in many patients of potentially serious complications known as late, or late onset complications. These include the specifically diabetic problems of nephropathy, retinopathy and neuropathy. These conditions are often referred to as microvascular complications even though microvascular alterations are not the only cause. Atherosclerotic disease of the large arteries, particularly the coronary arteries and the arteries of the lower extremities, may also occur.
Nephropathy develops in approximately 35% of IDDM patients, particularly in male patients and in those with onset of the disease before the age of 15 years. Diabetic nephropathy is characterized by persistent albuminuria secondary to glomerular capillary damage, a progressive reduction of the glomerular filtration rate and eventually, end stage renal failure.
The prevalence of diabetic retinopathy is highest among young-onset IDDM patients and it increases with the duration of the disease. Proliferative retinopathy is generally present in about 25% of the patients after 15 years duration and in over 50% after 20 years. The earliest lesion of diabetic retinopathy is a thickening of the capillary basement membrane, followed by capillary dilation and leakage and formation of microaneurysms. Subsequently, occlusion of retinal vessels occurs resulting in hypoperfusion of parts of the retina, oedema, bleeding and formation of new vessels as well as progressive loss of vision.
Diabetic neuropathy includes a wide variety of disturbances of somatic and autonomic nervous function. Sensory neuropathy may cause progressive loss of sensation or, alternatively, result in unpleasant sensations, often pain, in the legs or feet. Motor neuropathy is usually accompanied by muscle wasting and weakness. Nerve biopsies generally show axonal degeneration, demyelination and abnormalities of the vasa nervorum. Neurophysiological studies indicate reduced motor and sensory nerve conduction velocities. Autonomic neuropathy afflicts some 40% of the patients with IDDM of more than 15 years duration. It may evolve through defects in thermoregulation, impotence and bladder dysfunction followed by cardiovascular reflex abnormalities. Late manifestations may include generalized sweating disorders, postural hypotension, gastrointestinal problems and reduced awareness of hypoglycemia. The latter symptom has grave clinical implications.
A number of theories have been advanced with regard to possible mechanism(s) involved in the pathogenesis of the different diabetic complications, but this has not yet been fully elucidated. Metabolic factors may be of importance and it has been shown that good metabolic control is accompanied by significantly reduced incidence of complications of all types. Nevertheless, after 7-10 years of good metabolic control, as many as 15-25% of the patients show signs of beginning nephropathy, 10-25% have symptoms of retinopathy and 15-20% show delayed nerve conduction velocity indicating neuropathy. With longer duration of the disease the incidence of complications increases further. There is thus a significant clinical need for the control and management of these diabetic complications.
Proinsulin C-peptide is a part of the proinsulin molecule which, in turn, is a precursor to insulin formed in the beta cells of the pancreas. For a long time it was believed that C-peptide (known variously as C-peptide or proinsulin C-peptide) had no role other than as a structural component of proinsulin, facilitating correct folding of the insulin part. However, it has in more recent years been recognised that C-peptide has a physiological role as a hormone in its own right (Wahren et al., (2000), Am. J. Physiol. Endocrinol. Metab, 278, E759-E768). In diabetic patients, it alleviates renal dysfunction, improves blood flow in several tissues, ameliorates nerve functional impairments and is believed to delay or prevent the onset of late complications (Wahren et al., (2000) supra; Wahren and Johansson (1998), Horm. Metab. Res. 30, A2-A5). Indeed, C-peptide has been proposed for use in the treatment of diabetes in EP 132769 and in SE460334 for use in combination with insulin in the treatment of diabetes and prevention of diabetic complications.
Proinsulin, or large parts of it, is known in 37 different variants, representing 33 different species, ranging from Atlantic hagfish, Myxine glutinosa, to human. Whilst the insulin segments (i.e. the A and B chains of proinsulin) are well conserved between species, C-peptide is much more highly variable, showing not only sequence variation, but also several internal deletions, making the length of C-peptide variable (see FIG. 1).
Human C-peptide is a 31 amino acid peptide having the following sequence:
EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ.(SEQ ID. NO. 1)
C-peptide can be ascribed a tripartite overall structure, with more conserved N- and C-terminal segments and a more variable mid-sequence, or internal, portion. Thus, in the case of human C-peptide the N-terminal segment can be regarded as residues 1-12, the mid-portion as residues 13-25, and the C-terminal segment as residues 26-31.
The C-terminal pentapeptide fragment of C-peptide has been shown to have similar physiological and molecular effects to C-peptide itself, suggesting that this segment is an essential part of C-peptide (Wahren et al., 2000, supra; Rigler et al., 1999, PNAS USA 96, 13318-13323; Ohtomo et al., 1998, Diabetologia 41, 287-291; Pramanik et al., 2001, BBRC 284, 94-98; Shafqat et al, 2002, Cell Mol. Life. Sci., 59, 1185-1189). WO 98/13384 proposes the use of this C-terminal pentapeptide, and other C-terminally located peptide fragments of C-peptide in the treatment of diabetes and diabetic complications. The mid-sequence portion also has been shown to have molecular and physiological effects (see e.g. Ido et al. (1997, Science 277, 563-566) and Ohtomo et al., (1998), supra) and has also been proposed in WO 98/13384 to have clinical utility. Ido et al., have speculated that the mid-portion may mediate its effects through membrane interactions, although this is still to be confirmed and is not supported by other studies. The N-terminal segment of C-peptide although not active on its own, has also recently been shown to be functionally important and to contribute to C-peptide activity. Accordingly, various N-terminal fragments and derivatives of C-peptide, including in particular variants of C-peptide modified in the N-terminal region, have also been proposed in WO 2004/016647 to have therapeutic utility.
C-peptide thus appears to be a somewhat “complex” molecule, which may exert its effects by a variety of different mechanisms, including possibly via interaction with more than one receptor and/or more than one signalling pathway. Direct membranotropic mechanisms may also be involved although, as mentioned above, this is yet to be conclusively established. Thus, not only C-peptide itself (i.e. an intact native or wild-type C-peptide) but also various C-peptide fragments and modified variants or analogues thereof have therapeutic potential in the treatment and/or prevention of diabetes and/or diabetic complications. The use or potential use of all such C-peptides, and fragments and derivatives thereof is referred to herein as “C-peptide therapy”.
C-peptide is known, however, to have a relatively short half-life in the body (specifically in the circulation or plasma). The major degradation and removal of C-peptide takes place in the kidneys (Faber et al., Diabetes, 27, 207-209, 1978; Zavaroni et al., J. Clin. Endocrinol. Metab., 65, 494-498, 1987), although little is known in the prior art concerning the proteolytic enzymes involved and the mode of degradation.
Studies have shown that both C-peptide and its C-terminal pentapeptide are degraded in serum, with a longer half-life for intact C-peptide than for the C-terminal pentapeptide. Preliminary evidence suggests that the two peptides may be degraded in different ways in the serum. (Melles et al., Cell. Mol. Life. Sci., 60, 1019-1025, 2003).
The in vivo half life of C-peptide circulating in blood has been reported as approximately 30 minutes (as compared to 4-5 minutes for insulin), and that of the C-terminal pentapeptide is believed to be even shorter. Studies have shown that a dose of C-peptide injected into a rat would be expected to have disappeared entirely from circulation within 2-3 hours. Thus, in studying C-peptide physiological activity or in C-peptide therapy, it has been customary to administer C-peptide in several daily doses or to use a continuous dose. Similarly insulin, which is derived from the same prohormone (proinsulin) as C-peptide requires administration 3-5 times daily.
Hence, formulations of C-peptide which require fewer daily administrations would have clear clinical benefits. In WO 02/38129, a delayed release formulation of C-peptide is described, where C-peptide is present in an absorbable matrix designed to slow down the release of the peptide after administration to the patient.
The present invention is directed towards the aim of providing a longer lasting treatment and in particular towards providing a sustained release formulation of C-peptide. In this regard, the inventors have surprisingly found that C-peptide can form a gel under particular conditions. In particular, it has been found that a C-peptide composition may be transformed into a gellous state when combined with metal ions and/or by adjusting the pH of the composition. It is proposed that by using a C-peptide composition in such a gellous state (i.e. in the form of a gel) a sustained release formulation may be obtained, which allows for the sustained release of the C-peptide over a period of time after administration in vivo.
Thus, experiments have been conducted which show that such a gel composition may release both the C-peptide, and the metal ion (when present) over time in a manner controlled by the composition of the gel. This strongly indicates that the gel can be used as a sustained release formulation of C-peptide. The beneficial features of the present invention include the simplicity and the ease with which the gels can be made, the fact that few ingredients are required, and the long shelf life of the gels.